Project Summary/Abstract Because exosomes mirror the origin and status of the cells, the analysis of the content encapsulated in exosomes from biological fluids can reveal information relevant to human health and disease. However, due to the paucity of highly effective and rigorous exosome isolation methods, identifying exosome content carrying biological information remains challenging. To advance our understanding of exosomes from breast cancer cells, we developed an innovative exosome isolation technology. With this new tool, we will investigate the function of exosomes in early detection and staging of breast cancer. In Aim 1 we will expand our new isolation technology that reports exosome proteome and validate the functionality extensively. In Aim 2, we will investigate how the release of exosomes is modulated in breast cancer cells in in vitro cultures and cell-derived xenografts. Successful data collection and analysis from this proposed study will not only determine the applicability of this novel technique but will also uncover the association between exosomes and breast cancer cell states, thus providing molecular signatures that can potentially serve as biomarkers and therapeutic choices for breast cancer.